1. Field of the Invention
This invention relates to two alkylresorcinol synthase genes cloned from sorghum, the sorghum alkylresorcinol synthase 1 gene, ARS1, and the alkylresorcinol synthase 2 gene, ARS2; constructs containing the ARS1 gene or the ARS2 gene and its promoter; a vector containing a ARS1 or ARS2 gene; ARS1 and ARS2 protein; a method of making ARS1 and ARS2 protein; a method of transforming plants; and transgenic plants which express ARS1 or ARS2 resulting in the biosynthesis of alkylresorcinol precursors to sorgoleone in planta, RNAi constructs, and a method of blocking the production of sorgoleone through RNA interference.
2. Description of the Relevant Art
Allelopathy, a form of chemical warfare between plants, can be defined as the production and release of chemical substances by one species that inhibit the growth of another species (Inderjit and Duke. 2003. Planta 217:529-539; Weston and Duke. 2003. Crit. Rev. Plant Sci. 22:367-389). Allelopathic interactions have been proposed to have profound effects on the evolution of plant communities through the loss of susceptible species via chemical interference, and by imposing selective pressure favoring individuals resistant to inhibition from a given allelochemical (e.g., Schulz and Wieland. 1999. Chemoecology 9:133-141). Furthermore, allelopathic compounds released by grain crop species are thought to play a significant role in cover crops or within intercropping systems where they act as weed suppressants. Allelopathic compounds have been characterized in number of plants such as black walnut, wheat, rice, and sorghum (Bertin et al. 2003. Plant Soil 256: 67-83; Inderjit and Duke, supra; Duke at al. 2005. Outlooks Pest Management 16: 64-68).
Despite the ecological and agronomic importance of allelochemicals, relatively few pathways have been characterized in detail at the molecular level. One notable exception is the identification and characterization of all the genes encoding the enzymes responsible for the biosynthesis of the benzoxazinoid, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one in Zea mays (Frey et al. 1997. Science 277:696-699). Benzoxazinoids are thought to act as alleopathic chemicals in the rhizosphere, in addition to being defense compounds against microbial pathogens and insect herbivores (Sicker at al. 2000. Int. Rev. Cytol. 198:319-346; Friebe, A. 2001. J. Crop Prod. 4:379-400).
Sorgoleone, an allelochemical of particular interest to plant chemical ecology as well as agriculture, has only been found to be produced by members of the genus Sorghum (Czarnota at al. 2003b. J. Chem. Ecol. 29:2073-2083; Baerson et al. 2008b. Plant Signal Behav. 3:667-670). The term sorgoleone is most frequently used to describe the compound corresponding to the predominant congener identified in sorghum root exudates (Netzly et al. 1988. Weed Sci. 36:441-446; Kagan et al. 2003. J. Agric. Food Chem. 51: 7589-7595), 2-hydroxy-5-methoxy-3-[(Z,Z)-8′,11′,14′-pentadecatriene]-p-benzoquinone (FIG. 1), which has been estimated to account for between approximately 40-90% of the exudate material (w/w) in various accessions (e.g. Nimbal et al. 1996. J. Agric. Food Chem. 44: 1343-1347; Czarnota et al. 2001. Weed Technol. 15: 813-825; Baerson et al. 2008a. J. Biol. Chem. 283:3231-3247; Dayan et al. 2009. J. Exp. Bot. 60:2107-2117). The remaining exudate consists primarily of 4,6-dimethoxy-2-[(Z,Z)-8′,11′,14′-pentadecatriene]resorcinol(methoxy-dihydrosorgoleone), and sorgoleone congeners differing in the length or degree of saturation of the aliphatic side chain, and in the substitution pattern of the quinone ring (Erickson et al. 2001. J. Agric. Food Chem. 49: 5537-5542; Kagan et al., supra; Rimando et al. 2003. J. Nat. Prod. 66: 42-45; Dayan et al. 2009, supra). The fact that sorgoleone acts as a potent broad-spectrum inhibitor active against many agronomically important monocotyledonous and dicotyledonous weed species, exhibits a long half-life in soil, and appears to affect multiple targets in vivo (e.g., Netzly & Butler. 1986. Crop Sci. 26: 775-780; Einhellig and Souza. 1992. J. Chem. Ecol. 18: 1-11; Nimbal et al., supra; Rimando et al., 1998. J. Nat. Prod. 61: 927-930; Czarnota et al. 2001. Weed Technol. 15: 813-825; Bertin et al. 2003. Plant Soil 256:67-83; Duke, S. O. 2003. Trends Biotechnol. 21: 192-195) may make it promising for development as a natural product alternative to synthetic herbicides (Duke, supra).
The biosynthesis of sorgoleone is thought to occur exclusively in root hairs, which appear as cytoplasmically dense cells in sorghum, containing large osmiophilic globules presumably associated with sorgoleone rhizosecretion (Czarnota et al. 2001, supra; Czarnota et al. 2003a. Int. J. Plant Sci. 164:861-866). Prior labeling studies have indicated a polyketide origin for the quinone ring of sorgoleone (Fate and Lynn. 1996. J. Amer. Chem. Soc. 118:11369-11376; Dayan et al. 2003. J. Biol. Chem. 278: 28607-28611), thus lending support for the initial steps in the proposed biosynthetic pathway shown in FIG. 1, where 5-pentadecatrienyl resorcinol (5-[(8′Z,11′Z)-8′,11′,14′-pentadecatrienyl]resorcinol) is produced by a polyketide synthase enzyme accepting a 16:3Δ9,12,15 fatty acyl-CoA starter unit. A specific sub-class of type III polyketide synthases, referred to as alkylresorcinol synthases [first described in microorganisms—(Funa et al. 2006. Proc. Nat. Acad. Sci. USA 103:6356-6361; Funa et al. 2007. J. Biol. Chem. 282:14476-14481)], have been proposed to participate in the biosynthesis of plant alkylresorcinols such as 5-pentadecatrienyl resorcinol (Austin and Noel. 2003. Nat. Prod. Rep. 20: 79-110; Dayan et al. 2003, supra). Two S. bicolor fatty acid desaturases (designated DES2 and DES3) likely involved in the formation of the proposed 16:3Δ9,12,15 fatty acyl-CoA starter unit have recently been characterized (Pan et al. 2007. J. Biol. Chem. 282:4326-4335). Subsequent modification of the 5-pentadecatrienyl resorcinol intermediate is likely mediated by the AdoMet-dependent O-methyltransferase OMT3 (Baerson et al. 2008a. J. Biol. Chem. 283:3231-3247) and by unidentified hydroxylases (possibly P450 monooxygenases), yielding dihydrosorgoleone, which rapidly undergoes oxidation to the benzoquinone (FIG. 1).
Type III polyketide synthases, which have been identified in both plants and microorganisms, are involved in the biosynthesis of a wide array of natural products, including flavonoids derived from the key intermediate 2′,4,4′,6′-tetrahydroxychalcone synthesized by the enzyme chalcone synthase (CHS; Austin and Noel, supra). These enzymes occur as homodimers possessing subunits between 40-45 kDa in size, and catalyze iterative decarboxylative condensation reactions, typically using malonyl-CoA extender units. Type III PKSs from various sources can differ in the types of starter units accepted, the number of condensation steps performed, and the type of intramolecular cyclization reaction performed, all of which contribute to the diversity of compounds produced by these enzymes (Austin and Noel, supra; Khosla et al., 1999. Annu. Rev. Biochem. 68:219-253). For example, the closely-related CHS and stilbene synthase (STS) type III enzymes both catalyze the formation of identical tetraketide intermediates from p-coumaryl-CoA, yet form different products due to cyclization occurring via a C6→C1 Claisen condensation for CHS, and a C2→C7 aldol condensation for STS-type enzymes (Tropf et al. 1994. J. Mol. Evol. 38:610-618). Alkylresorcinol synthases, which produce 5-alkylresorcinols from fatty acyl-CoA starter units, also use a STS-type cyclization mechanism, and with specific acyl-CoA starters may also generate pyrone by-products via intramolecular C5 oxygen→C1 lactonization (Funa et al. 2006, supra; Funa et al. 2007, supra; Funabashi at al. 2008. J. Biol. Chem. 283: 13983-13991; Goyal et al. 2008. J. Struct. Biol. 162:411-421).
Alkylresorcinols are members of an extensive family of compounds possessing varied bioactivities and biological roles referred to as phenolic lipids, which are thought to be derived predominantly from polyketide-associated pathways (Austin and Noel, supra). Sorgoleone represents one of the more extensively-studied phenolic lipids identified in plants; other important examples include urushiol, an allergen from poison ivy (Toxicodendron radicans), anacardic acid, an anti-feedant found in several dicotyledonous species such as cashew (Anacardium occidentale), as well as the alkylresorcinol phytoanticipins found throughout the Poaceae (grass) family (Kozubek and Tyman. 1999. Chem. Rev. 99:1-26; Kozubek et al., 2001. Cell. Mol. Biol. Lett. 6:351-355). Plant-derived phenolic lipids have also been used by industry, for example in manufacturing of formaldehyde-based polymers and in lacquering processes (Kozubek and Tyman, supra).
Prior studies on type III PKS-like sequences from S. bicolor have involved the characterization of 8 sequences (designated CHS1-8) obtained from genomic library screens and analysis of expressed sequence tags (Lo et al. 2002. Physiol. Mol. Plant. Path. 61:179-188; Yu at al. 2005. Plant Physiol. 138:393-401). Recombinant enzyme studies have identified CHS8 as an STS and CHS2 as a typical CHS-type enzyme, and it is has been proposed that CHS1, 3, 4, 5, 6 and 7 also represent CHS-type enzymes given their high degree of sequence identity (≧97.5%) with CHS2 (Christine et al., supra).
Relatively little functional data exists concerning the genes and corresponding enzymes involved in the biosynthesis of alkylresorcinols in higher plants, thus new tools for exploring related pathways are needed, particularly in the Poaceae family where the occurrence of presumed phytoanticipin alkyresorcinols is widespread. Here, we have cloned and characterized two paralogous alkylresorcinol synthases (ARS) from S. bicolor (genotype BTx623), designated ARS1 and ARS2, important for the biosynthesis of the 5-pentadecatrienyl resorcinol precursor to sorgoleone.